Dithioethanol (DTE)-Conjugated Deoxyribose Cyclic Dinucleotide Prodrugs (DTE-dCDNs) as STING Agonist

To improve the chemical regulation on the activity of cyclic dinucleotides (CDNs), we here designed a reduction-responsive dithioethanol (DTE)-based dCDN prodrug 9 (DTE-dCDN). Prodrug 9 improved the cell permeability with the intracellular levels peaking in 2 h in THP-1 cells. Under the reductive substance such as GSH or DTT, prodrug 9 could be quickly decomposed in 30 min to release the parent dCDN. In THP1-Lucia cells, prodrug 9 also retained a high bioactivity with the EC50 of 0.96 μM, which was 51-, 43-, and 3-fold more than the 2′,3′-cGAMP (EC50 = 48.6 μM), the parent compound 3′,3′-c-di-dAMP (EC50 = 41.3 μM), and ADU-S100 (EC50 = 2.9 μM). The high bioactivity of prodrug 9 was validated to be highly correlated with the activation of the STING signaling pathway. Furthermore, prodrug 9 could also improve the transcriptional expression levels of IFN-β, CXCL10, IL-6, and TNF-α in THP-1 cells. These results will be helpful to the development of chemically controllable CDN prodrugs with a high cellular permeability and potency.


Introduction
The cyclic GMP-AMP synthase (cGAS)-STING pathway has emerged as an important intrinsic tumor-sensing mechanism [1,2].Tumor-derived DNA activates cGAS to produce 2 ,3 -cGAMP, the endogenous ligand of STING, resulting in the downstream signaling cascade via the recruitment of threonine-protein kinase (TBK1), phosphorylation of the interferon regulatory transcription factor IRF3, and production of type I interferon (IFN), and other proinflammatory cytokines.In parallel, STING and TBK1 also interact with the inhibitors of κB kinases (IKKs), and then release the transcription factor NF-κB from its inhibitor, thus allowing the transcription of proinflammatory cytokines and chemokines such as tumor necrosis factor-α (TNFα) and interleukin 6 (IL6), via the nuclear factor κ-lightchain-enhancer of the activated B cells (NF-κB) pathway [3,4].These events can selectively stimulate the cross-presentation of tumor antigens and mobilization of tumor-specific CD8 + T cells, which prime the adaptive immune response against tumors [5,6].Accordingly, STING has been widely investigated as a therapeutic target for the treatment of infectious diseases, cancer immunotherapy, and autoimmunity, as well as vaccine adjuvants [7][8][9][10].
Owing to some limitations of endogenous cyclic dinucleotides (CDNs), such as rapid clearance and poor membrane permeability, there has been an increased interest in identifying new STING agonists with improved drug-like properties compared to natural STING ligands [11][12][13].However, the cell delivery and bioactivity of CDNs were still unmet in clinical trials, which need further structure optimization.
The finding and design of different structures of CDNs were the main goals to enhance bioactivity, selectivity, stability, and safety [14][15][16].In general, CDNs are polar molecules bearing two negative charges on the phosphoric oxygen atoms, and their cellular uptake requires the presence of transporters since the free diffusion of polar molecules via the lipid cell membrane is limited [17].For a successful drug design, the negative charges of CDNs need to be masked to increase their ability to enter cells via diffusion through the cell membrane.Prodrug strategies were developed to improve the transport of polar nucleoside phosphates and phosphonates across the cell membrane to cells, where the biolabile protecting groups are enzymatically or chemically cleaved in an intracellular environment and the active parent species is released [18][19][20][21].
In our previous work, we have designed esterase-responsive SATE-dCDN prodrugs with a high cell uptake efficiency and high bioactivity [21].Since the bioactivity of SATE-dCDN strictly relies on the abundance of cellular esterase, the programable regulation of their activity was mostly limited in multiple tissues, which will add the risk of systemic toxicity.To further alleviate the potential risk, a naturally regulated and selective release strategy could be introduced.Reduced glutathione (GSH) is an important substance that determines the redox environment in organisms.It is worth noting that the concentration of GSH in tumor tissues was at least four times higher than that in normal tissues, and the intracellular concentration of GSH was up to 2-10 mmol/L, which is an ideal trigger for drug delivery [22].
Considering that disulfide bond is one of the most widely used redox-responsive connecting arms [23][24][25], in this study, we designed the dithioethanol (DTE)-based dCDN prodrug 9 (DTE-dCDN) to test cell uptake, controllable release, cellular bioactivity, and toxicity.For prodrug 9, when the DTE group masked the negatively charged phosphodiester group of dCDN, it helped CDN cross the cell membrane easily.Once these prodrugs enter the cell, the DTE group was then degraded, which was initiated by reductive reagents.As the disulfide bridge broke, the unstable intermediate with O-2-mercaptoetnylphosphotriester would decompose spontaneously via intermolecular nucleophilic displacement into the corresponding phosphodiester structure dCDN (Figure 1).Immediately after that, the free dCDN would stimulate the cGAS-STING pathway to induce the production of type I IFNs and proinflammatory cytokines.The design and bioactivity evaluation of 9 will provide an effective approach to chemically regulate the activity of CDNs.
enhance bioactivity, selectivity, stability, and safety [14][15][16].In general, CDNs are polar molecules bearing two negative charges on the phosphoric oxygen atoms, and their cellular uptake requires the presence of transporters since the free diffusion of polar molecules via the lipid cell membrane is limited [17].For a successful drug design, the negative charges of CDNs need to be masked to increase their ability to enter cells via diffusion through the cell membrane.Prodrug strategies were developed to improve the transport of polar nucleoside phosphates and phosphonates across the cell membrane to cells, where the biolabile protecting groups are enzymatically or chemically cleaved in an intracellular environment and the active parent species is released [18][19][20][21].
In our previous work, we have designed esterase-responsive SATE-dCDN prodrugs with a high cell uptake efficiency and high bioactivity [21].Since the bioactivity of SATE-dCDN strictly relies on the abundance of cellular esterase, the programable regulation of their activity was mostly limited in multiple tissues, which will add the risk of systemic toxicity.To further alleviate the potential risk, a naturally regulated and selective release strategy could be introduced.Reduced glutathione (GSH) is an important substance tha determines the redox environment in organisms.It is worth noting that the concentration of GSH in tumor tissues was at least four times higher than that in normal tissues, and the intracellular concentration of GSH was up to 2-10 mmol/L, which is an ideal trigger for drug delivery [22].
Considering that disulfide bond is one of the most widely used redox-responsive connecting arms [23][24][25], in this study, we designed the dithioethanol (DTE)-based dCDN prodrug 9 (DTE-dCDN) to test cell uptake, controllable release, cellular bioactivity, and toxicity.For prodrug 9, when the DTE group masked the negatively charged phosphodiester group of dCDN, it helped CDN cross the cell membrane easily.Once these prodrugs enter the cell, the DTE group was then degraded, which was initiated by reductive reagents.As the disulfide bridge broke, the unstable intermediate with O-2mercaptoetnylphosphotriester would decompose spontaneously via intermolecular nucleophilic displacement into the corresponding phosphodiester structure dCDN (Figure 1).Immediately after that, the free dCDN would stimulate the cGAS-STING pathway to induce the production of type I IFNs and proinflammatory cytokines.The design and bioactivity evaluation of 9 will provide an effective approach to chemically regulate the activity of CDNs.

Synthesis of Prodrug 9
Prodrug 9 was synthesized, as shown in Scheme 1. Compound 3 was obtained by coupling deoxynucleoside phosphoramidite 2 and 3-hydroxypropionitrile with the activator ETT in MeCN and then oxidized with TBHP.Only one cyanoethyl group for compound 3 was removed when treated with the tBuNH2-MeCN mixture, and then reacted with compound 1 with MSNT in anhydrous pyridine to form the intermediate 4 The cyanoethyl group for compound 4 was removed under the t-BuNH2/MeCN mixture to achieve intermediate 5 without further purification.The removal of the 5′-DMTr protecting groups of compounds 4 with a solution of 6% dichloroacetic acid (DCA) in

Synthesis of Prodrug 9
Prodrug 9 was synthesized, as shown in Scheme 1. Compound 3 was obtained by coupling deoxynucleoside phosphoramidite 2 and 3-hydroxypropionitrile with the activator ETT in MeCN and then oxidized with TBHP.Only one cyanoethyl group for compound 3 was removed when treated with the tBuNH 2 -MeCN mixture, and then reacted with compound 1 with MSNT in anhydrous pyridine to form the intermediate 4. The cyanoethyl group for compound 4 was removed under the t-BuNH 2 /MeCN mixture to achieve intermediate 5 without further purification.The removal of the 5 -DMTr protecting groups of compounds 4 with a solution of 6% dichloroacetic acid (DCA) in dichloromethane provided the intermediate 6.Subsequently, a combination of 5 and 6 in the presence of coupling reagent MSNT in pyridine and treatment with 6% DCA solution could generate the corresponding linear dinucleotide 7.After removing the cyanoethyl with the t-BuNH 2 /MeCN mixture, the cyclization was achieved with MSNT in pyridine to obtain the cyclic dinu-cleotide phosphotriester 8, which was then treated with 10% diisopropylamine (DIA) in methanol to achieve the DTE-dCDN prodrug 9 in a 12% overall yield.

Fast Uptake of DTE-dCDN Prodrug into THP-1 Cells
To determine the cell uptake of the DTE-dCDN prodrug, prodrug 9 and its parent dCDN were prepared.Prodrug 9 entered THP-1 cells rapidly and was efficiently metabolized to its parent dCDN 3′,3′-c-di-dAMP as determined via liquid chromatograph MS/MS analysis.As shown in Figure 2, the intracellular levels of prodrug 9 peaked in 2 h but then rapidly decreased, the intracellular levels of the parent dCDN 3′,3′-c-di-dAMP peaked in 6 h, while the intracellular levels of the parent dCDN 3′,3′-c-di-dAMP were not detectable within 6 h when the THP-1 cells were treated with 3′,3′-c-di-dAMP even at a 10 × higher concentration than was used in the case of prodrug 9. Contrary to the rapid entry of the prodrug, the parent CDN was barely detectable in cells after a 6 h incubation, and its intracellular concentration increased linearly for up to 24 h.These results indicated that the DTE-dCDN prodrug improved the cell permeability and could be quickly decomposed to release the parent dCDN in cell cytoplasm.

Fast Uptake of DTE-dCDN Prodrug into THP-1 Cells
To determine the cell uptake of the DTE-dCDN prodrug, prodrug 9 and its parent dCDN were prepared.Prodrug 9 entered THP-1 cells rapidly and was efficiently metabolized to its parent dCDN 3 ,3 -c-di-dAMP as determined via liquid chromatograph MS/MS analysis.As shown in Figure 2, the intracellular levels of prodrug 9 peaked in 2 h but then rapidly decreased, the intracellular levels of the parent dCDN 3 ,3 -c-di-dAMP peaked in 6 h, while the intracellular levels of the parent dCDN 3 ,3 -c-di-dAMP were not detectable within 6 h when the THP-1 cells were treated with 3 ,3 -c-di-dAMP even at a 10× higher concentration than was used in the case of prodrug 9. Contrary to the rapid entry of the prodrug, the parent CDN was barely detectable in cells after a 6 h incubation, and its intracellular concentration increased linearly for up to 24 h.These results indicated that the DTE-dCDN prodrug improved the cell permeability and could be quickly decomposed to release the parent dCDN in cell cytoplasm.

DTE-dCDN Prodrug Showed Reduction-Dependent Release of Parent dCDN
To analyze the reduction dependency, we performed the in vitro cleavage test by incubating prodrug 9 with a reductive substance such as GSH or DTT and cell lysates at 37 °C and then determined the change with HPLC.As shown in Figure 3, the elution time

DTE-dCDN Prodrug Showed Reduction-Dependent Release of Parent dCDN
To analyze the reduction dependency, we performed the in vitro cleavage test by incubating prodrug 9 with a reductive substance such as GSH or DTT and cell lysates at 37 • C and then determined the change with HPLC.As shown in Figure 3, the elution time of prodrug 9 and its parent dCDN 3 ,3 -c-di-dAMP were 6.0 min and 1.2 min, respectively.As the reductive substance or cell lysates were added, the peak for prodrug 9 disappeared and the new peaks at 1.2 min appeared in 0.5 h and 2 h, respectively.The released parent dCDN was also confirmed with 3 ,3 -c-di-dAMP through UPLC-MS (Figure S1).The results indicated that the DTE-dCDN prodrug 9 could be quickly decomposed to release the parent dCDN in a reductive substance such as DTT or GSH.

DTE-dCDN Prodrug Showed Reduction-Dependent Release of Parent dCDN
To analyze the reduction dependency, we performed the in vitro cleavage test by incubating prodrug 9 with a reductive substance such as GSH or DTT and cell lysates at °C and then determined the change with HPLC.As shown in Figure 3, the elution time of prodrug 9 and its parent dCDN 3′,3′-c-di-dAMP were 6.0 min and 1.2 min, respectively.As the reductive substance or cell lysates were added, the peak for prodrug 9 disappeared and the new peaks at 1.2 min appeared in 0.5 h and 2 h, respectively.The released parent dCDN was also confirmed with 3′,3′-c-di-dAMP through UPLC-MS (Figure S1).The results indicated that the DTE-dCDN prodrug 9 could be quickly decomposed to release the parent dCDN in a reductive substance such as DTT or GSH.

Prodrug 9 Relied on STING-Dependent Signal Transduction for the IFN-β Activation
To demonstrate the dependence of STING on the activity of prodrug 9, we incubated THP1-Lucia cells with prodrug 9 alone or pretreated with the STING and TBK1 inhibitor to measure the luciferase activity.The endogenous 2′,3′-cGAMP was set as the positive control group.H-151 [26] and BX795 [27] were selected to use as the STING inhibitor and TBK1 inhibitor, respectively.As shown in Figure 6, the inhibition of the downstream effector STING and TBK1, with H-151 and BX795, could block the induction of the luciferase expression by prodrug 9, which was in accordance with the test of the 2′,3′-cGAMP.The results indicated that prodrug 9 could stimulate the IFN-β promoter responsive luciferase expression through the STING signaling pathway.

Prodrug 9 Relied on STING-Dependent Signal Transduction for the IFN-β Activation
To demonstrate the dependence of STING on the activity of prodrug 9, we incubated THP1-Lucia cells with prodrug 9 alone or pretreated with the STING and TBK1 inhibitor to measure the luciferase activity.The endogenous 2 ,3 -cGAMP was set as the positive control group.H-151 [26] and BX795 [27] were selected to use as the STING inhibitor and TBK1 inhibitor, respectively.As shown in Figure 6, the inhibition of the downstream effector STING and TBK1, with H-151 and BX795, could block the induction of the luciferase expression by prodrug 9, which was in accordance with the test of the 2 ,3 -cGAMP.The results indicated that prodrug 9 could stimulate the IFN-β promoter responsive luciferase expression through the STING signaling pathway.

Prodrug 9 Relied on STING-Dependent Signal Transduction for the IFN-β Activation
To demonstrate the dependence of STING on the activity of prodrug 9, we incubated THP1-Lucia cells with prodrug 9 alone or pretreated with the STING and TBK1 inhibitor to measure the luciferase activity.The endogenous 2′,3′-cGAMP was set as the positive control group.H-151 [26] and BX795 [27] were selected to use as the STING inhibitor and TBK1 inhibitor, respectively.As shown in Figure 6, the inhibition of the downstream effector STING and TBK1, with H-151 and BX795, could block the induction of the luciferase expression by prodrug 9, which was in accordance with the test of the 2′,3′-cGAMP.The results indicated that prodrug 9 could stimulate the IFN-β promoter responsive luciferase expression through the STING signaling pathway.
In order to validate the STING activation in other cells like tumor cells, we chose colon cancer cells CT-26 as a model to investigate the effect of these compounds on the STING activation-mediated IFN-β gene expression.Colon cancer cells CT-26 showing the extremely low expression of STING proteins are usually used as a tumor model to verify the antitumor immunotherapeutic effect in mice.Under the treatment of prodrug 9 and CDNs, the mRNA expression levels of IFN-β were also assessed via real-time quantitative PCR (qPCR).As seen in Figure S2, all the compounds including ADU-S100 exhibited no significant induction on the transcription of IFN-β in CT-26 cells, which was contrary to the high bioactivity in THP-1 cells.It suggested that prodrug 9 exhibited its bioactivity relying on the normal STING expression as seen in THP-1 cells.
the antitumor immunotherapeutic effect in mice.Under the treatment of prodrug 9 and CDNs, the mRNA expression levels of IFN-β were also assessed via real-time quantitative PCR (qPCR).As seen in Figure S2, all the compounds including ADU-S100 exhibited no significant induction on the transcription of IFN-β in CT-26 cells, which was contrary to the high bioactivity in THP-1 cells.It suggested that prodrug 9 exhibited its bioactivity relying on the normal STING expression as seen in THP-1 cells.Next, we employed the ELISA method to examine the actual protein levels of IFN-β in the THP-1 cells treated with prodrug 9, 3 ,3 -c-di-dAMP, 2 ,3 -cGAMP, and ADU-S100.As shown in Figure S3, prodrug 9 induced the protein levels of IFN-β by approximately 66-, 4.3-, and 5.6-fold compared to 2 ,3 -cGAMP, 3 ,3 -c-di-dAMP, and ADU-S100.The results of the ELISA are consistent with those from the qPCR analysis.Hence, prodrug 9 could induce the STING signaling pathway to increase the transcriptional and protein expression levels of IFN-β and other proinflammatory cytokines.

Cell Viability of Prodrug 9 in THP-1 Cells and HEK293T Cells
To evaluate the safety of the designed prodrug, cell counting kit-8 (CCK8) was used to quantitatively assess the cell viability of the DET-dCDN prodrug 9, 3 ,3 -c-di-dAMP, and 2 ,3 -cGAMP.The CCK8 assay revealed that prodrug 9 has no significant influence on the growth of THP-1 and HEK293T cells (Figure 8).It suggested that prodrug 9 showed an undetectable cytotoxicity to THP-1 cells and HEK293T cells.
could induce the STING signaling pathway to increase the transcriptional and protein expression levels of IFN-β and other proinflammatory cytokines.

Cell Viability of Prodrug 9 in THP-1 Cells and HEK293T Cells
To evaluate the safety of the designed prodrug, cell counting kit-8 (CCK8) was used to quantitatively assess the cell viability of the DET-dCDN prodrug 9, 3′,3′-c-di-dAMP, and 2′,3′-cGAMP.The CCK8 assay revealed that prodrug 9 has no significant influence on the growth of THP-1 and HEK293T cells (Figure 8).It suggested that prodrug 9 showed an undetectable cytotoxicity to THP-1 cells and HEK293T cells.

Chemistry
Unless otherwise specified, all solvents and reagents were purchased from commercial sources and used without further purification.Reactions were monitored by TLC on silica gel GF254 with detection under UV light.Column chromatography was performed using 300-400 mesh or 200-300 mesh silica gel.NMR spectra were recorded on Bruker AVANCE 400 M instrument (Chemical characterization of 1 H, 13 C, 31 P NMR and HPLC spectra can be seen in pages S6-S15 in Supplementary Materials).Chemical shifts (δ) were reported in ppm downfield from an internal TMS standard, and J values were given in Hz.The following abbreviations were used to explain the multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet).The number of protons (n) for a

Chemistry
Unless otherwise specified, all solvents and reagents were purchased from commercial sources and used without further purification.Reactions were monitored by TLC on silica gel GF254 with detection under UV light.Column chromatography was performed using 300-400 mesh or 200-300 mesh silica gel.NMR spectra were recorded on Bruker AVANCE 400 M instrument (Chemical characterization of 1 H, 13 C, 31 P NMR and HPLC spectra can be seen in pages S6-S15 in Supplementary Materials).Chemical shifts (δ) were reported in ppm downfield from an internal TMS standard, and J values were given in Hz.The following abbreviations were used to explain the multiplicities: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet).The number of protons (n) for a given resonance were indicated as nH.HRMS (MALTI-TOF) was obtained from Varian 7.0T FTMS.HPLC was achieved using Agilent 1260 (Agilent Technologies, Santa Clara, CA, USA).Preparative HPLC was performed on an Agela OCTOPUS purification system with monitoring at 254 nm on an ASB C18 column (10 µm OBD, 21.2 × 250 mm) (Agela Technologies, Tianjin, China) using gradients of H 2 O and MeCN at a flow rate of 10 mL/min.Purity of all final compounds tested in biological assays was determined to be >95% by HPLC analysis.The following analytical method was used to determine the chemical purity of the final compounds: HPLC, Agilent 1260, 10 mM TEAA buffer (mobile phase A), MeCN (mobile phase B), column Agilent ZORABX SB-C18 5 µm [4.6 × 150 mm], column temperature 25 • C, 1 mL/min, 254 nm.The gradient was as follows: 0-2 min: 98% A/2% B; 2-6 min: 98% A/2% B to 100% B; 6-10 min: 100% B; 10-13 min: 100% B to 98% A/2% B; 13-15 min: 98% A/2% B. 3 ,3 -c-di-AMP and 2 ,3 -cGAMP were prepared according to the literature procedures [15].ADU-S100 was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA, Cat.No: HY-12885B).
Synthesis of Compound 3. Deoxynucleoside phosphoramidite (2) (0.99 g, 1.12 mmol), 3-Hydroxypropionitrile (85 mg, 1.20 mmol), and ETT (440 mg, 3.38 mmol) were dissolved in dry MeCN (20 mL).The mixture was stirred at room temperature for 1 h under Ar atmosphere.An amount of 1 mL TBHP (5.5 M in decane) was added for another 40 min.Afterwards, the solvent was removed under reduced pressure and the residue was resolved with 20 mL DCM.The reaction was washed with a saturated aqueous solution of NaHCO 3 and extracted with DCM (2 × 20 mL).The organic phase was combined, washed with NaCl saturated solution, dried over anhydrous Na 2 SO 4 , filtered, and concentrated.The residue was purified via chromatography on silica gel (DCM: MeOH = 100:1~50:1) to give compound 3 as a white solid (890 mg, yield 91.0%).
3: Synthesis of Compound 4. In a 50 mL round flask, compound 3 (1.20 g, 1.37 mmol) was dissolved in 10 mL MeCN and 3 mL tert-butylamine and then stirred at room temperature for 20 min.The solvent was evaporated under reduced pressure.Compound 1 (260 mg, 1.37 mmol) and MSNT (1.22 g, 4.12 mmol) were added into the flask, the mixture was dissolved in 10 mL anhydrous pyridine, and stirred at room temperature under Ar atmosphere overnight.Several drops of water were added into the flask to stop the reaction.Afterwards, the solvent was removed under reduced pressure and resolved with 20 mL DCM again.Oxalic acid aqueous solution was added into the mixture.The organic phase was separated, and the water phase was extracted with DCM (2 × 50 mL).The organic phase was combined and washed with water twice (2 × 50 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated.The residue was purified via chromatography on silica gel (DCM: MeOH = 100:1~50:1) to give compound 4 as a white solid (946 mg, yield 69.8%).
4: Prodrug 9 release parent drug initiated by cell lysates, DTT, and GSH.HEK 293T cells were plated in a 6-well plate at a density of 10 6 cell per well.After 48 h, the culture media was removed and washed with 500 µL PBS buffer.Cells were collected into 1.5 mL microtubes.An amount of 200 µL weak RIPA lysis buffer containing 1 mM protease inhibitor PMSF (LEAGENE, Trenton, NJ, USA) was added to each tube for 30 min.After centrifugation at 13,000 rpm for 10 min, the supernatant (180 µL) was transferred into a new tube.An amount of 100 µM prodrug 9 was added into the cell lysate (60 µL) in an air-bath incubator at 37 • C. Aliquots of the reaction mixture were collected at various time points (0.5 h and 2 h) and stopped with 50 µL MeCN and 50 µL H 2 O followed by centrifugation at 13,000 rpm for 5 min.Finally, 10 µL of each aliquot was injected directly into the HPLC (Agilent 1260 equipped with a UV detector; column Agilent ZORABX SB-C18 5 µm [4.6 × 150 mm]; detection at 254 nm; column temperature 25 • C) for analysis.Amounts of 10 mM TEAA buffer (solvent A) and MeCN (solvent B) were used as the mobile phase with a flow rate of 1 mL/min.The gradient was set as the following: 0-2 min: 98% A/2% B; 2-6 min: 98% A/2% B to 100% B; 6-10 min: 100% B; 10-13 min: 100% B to 98% A/2% B; 13-15 min: 98% A/2% B.
Amounts of 10 mM DTT or GSH were added into prodrug 9 (100 µM) in an air-bath incubator at 37 • C for 0.5 h.Afterwards, 10 µL of each mixture was injected into HPLC for analysis.The HPLC methodology was the same as above.
In vitro serum stability assay.Each compound (100 µM) was incubated at 37 • C in the reaction buffer including 20% fetal bovine serum (FBS, GIBCO, Thermo Fisher Scientific, Waltham, MA, USA), 10 mM PBS (pH 7.4), and 1 mM MgCl 2 .At various times, aliquots of the reaction mixture were collected and stopped by adding 50 µL MeCN and diluted with 50 µL water.The reaction mixture was then centrifuged at 10,000 rpm for 5 min, leaving 100 µL supernatant for the HPLC assay.An amount of 10 µL of each aliquot was injected directly into HPLC (Agilent 1260 Infinity HPLC equipped with a UV detector; column Agilent ZORABX SB-C18 5 µm [4.6 × 150 mm; detection at 254 nm; column temperature 25 • C] for analysis.Amounts of 10 mM TEAA buffer (solvent A) and MeCN (solvent B) were used as the mobile phase with a flow rate of 1 mL/min.The gradient was set as the following: 0-2 min: 98% A/2% B; 2-6 min: 98% A/2% B to 100% B; 6-10 min: 100% B; 10-13 min: 100% B to 98% A/2% B; 13-15 min: 98% A/2% B. The % remaining of the test compounds after incubation in the serum was then calculated.
THP1-Lucia cell-based reporter assay.THP1-Lucia cells were seeded into a 24-well plate at a density of 5 × 10 4 cells/well.After 16 h of incubation at 37 • C in a 5% CO 2 atmosphere, serially diluted compounds were added to cell cultures and incubated for 24 h at 37 • C in a 5% CO 2 atmosphere.Finally, the medium was collected to determine the luciferase activity using QUANTI-Luc (InvivoGen, San Diego, CA, USA) according to the manufacturer s instruction to calculate the EC 50 values of the tested compounds.The results are expressed as the mean ± SD from three independent experiments.Emax values were normalized to the response induced by 2 ,3 -cGAMP in each assay and expressed as a mean percentage ± SD from three independent experiments.The THP1-Lucia cell line was obtained from Prof. Junmin Quan from Peking University (Beijing, China) [21].
Real-time quantitative PCR (RT-qPCR).The mRNA expression levels of diverse cytokines were measured using quantitative RT-PCR (RT-qPCR) assays.THP-1 cells or CT26 cells were incubated with the compounds for 4 h, subsequently harvested for RNA isolation according to the manufacturer's instructions of RNApure Tissue&Cell Kit (DNase I) (CWBIO, Cambridge, MA, USA catalogue no.CW0560S).Next, 1 µg RNA was reversely transcribed to cDNA by TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen, Beijing, China).To examine the mRNA levels, real-time PCR was performed in a CFX96 Real-Time PCR system (Bio-RAD, Hercules, CA, USA) using PerfectStart Green qPCR Super Mix (TransGen) and primers (Sangon Biotech, Shanghai, China).The RT-qPCR primers used in this study are listed in Table S1.The expression level was calculated according to the formula (2 −∆∆Ct ) using the GAPDH or β-actin as the internal reference gene and the relative gene expression fold was normalized

Figure 3 .
Figure 3. Parent dCDNs release in GSH, DTT (a), and cell lysates (b).An amount of 100 µM prodrug was added into the 10 mM GSH, 10 mM DTT, and cell lysates for various times (0.5 h and 2 h) at °C and analyzed with HPLC (Agilent 1260, Santa Clara, CA, USA).

Figure 3 .
Figure 3. Parent dCDNs release in GSH, DTT (a), and cell lysates (b).An amount of 100 µM prodrug 9 was added into the 10 mM GSH, 10 mM DTT, and cell lysates for various times (0.5 h and 2 h) at 37 • C and analyzed with HPLC (Agilent 1260, Santa Clara, CA, USA).

Figure 5 .
Figure 5.The luciferase-based assay in the THP1-Lucia reporter cell line.(a) EC50 values represent the mean (±SD) of three independent experiments.(b) Emax values represent the mean (±SD) of three independent experiments.

Figure 5 .
Figure 5.The luciferase-based assay in the THP1-Lucia reporter cell line.(a) EC 50 values represent the mean (±SD) of three independent experiments.(b) E max values represent the mean (±SD) of three independent experiments.
Subsequently, a combination of 5 and 6 in the presence of coupling reagent MSNT in pyridine and treatment with 6% DCA solution could generate the corresponding linear dinucleotide 7.After removing the cyanoethyl with the t-BuNH2/MeCN mixture, the cyclization was achieved with MSNT in pyridine to obtain the cyclic dinucleotide phosphotriester 8, which was then treated with 10% diisopropylamine (DIA) in methanol to achieve the DTE-dCDN prodrug 9 in a 12% overall yield.